1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
2.1. THE HUMAN IMMUNODEFICIENCY VIRUS
2.2. HIV TREATMENT
2.3. LIPOPOLYSACCHARIDES
3. SUMMARY OF THE INVENTION
3.1. DEFINITIONS
4. DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. LIPOPOLYSACCHARIDE VARIANTS AND DERIVATIVES AND ANALOGS THEREOF WITH REDUCED OR ABSENT PYROGENICITY
5.2. SYNTHESIS AND ISOLATION OF LIPOPOLYSACCHARIDES
5.2.1 PURIFICATION OF LIPOPOLYSACCHARIDES FROM MICROORGANISMS
5.2.1.1 SOURCES OF LIPOPOLYSACCHARIDES
5.2.1.2 ISOLATION OF LIPOPOLYSACCHARIDES
5.2.2 SYNTHESIS OF LIPOPOLYSACCHARIDES
5.3 THERAPEUTIC USES
5.4 COMBINATION THERAPY
5.5 DEMONSTRATION OF THERAPEUTIC UTILITY
5.4.1 DETERMINING THE PYROGENICITY OF THE PREPARATION
5.4.2 DETERMINING THE ANTI-HIV ACTIVITY OF THE PREPARATION
5.6 THERAPEUTIC COMPOSITIONS AND METHODS OF ADMINISTRATION
6. EXAMPLE: NON-PYROGENIC LPS STIMULATES xcex2 CHEMOKINE SECRETION IN PBMC
6.1 MATERIALS AND METHODS
6.2 RESULTS
6.3 DISCUSSION
7. EXAMPLE: INHIBITION OF HIV-1 REPLICATION IN HUMAN PBMC-DERIVED MONOCYTES BY NON-PYROGENIC LPS
7.1 METHOD
7.2 RESULTS
8. EXAMPLES: SYNTHETIC LIPID IVA SUPPRESSES HIV REPLICATION WITHOUT INDUCING MEASURABLE LEVELS OF xcex2 CHEMOKINES
8.1 METHODS AND RESULTS
9. EXAMPLES: NON-PYROGENIC LPS SUPPRESSES HIV REPLICATION WITHOUT DISPLAYING LPS ANTAGONIST ACTIVITY
9.1 METHODS AND RESULTS
The present invention relates to lipopolysaccharide (LPS) or lipid A variants, derivatives, and analogs with non-pyrogenic and non-endotoxic properties as well as methods for treatment and prevention of immunodeficiency virus infection, in particular HIV infection, using these LPS or lipid A variants and analogs and derivatives. The present invention also relates to LPS and lipid A antagonists and their use as therapeutics in the treatment and prevention of HIV infection. The LPS and lipid A variants, derivatives, and analogs of the present invention preferably induce the secretion of xcex2 chemokines but exhibit decreased induction relative to LPS and lipid A of secretion of proinflammatory cytokines, such as IL-1xcex2, IL-6 and TNF-xcex1. The present invention further relates to pharmaceutical compositions for the treatment and prevention of HIV infection.
2.1. THE HUMAN IMMUNODEFICIENCY VIRUS
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F., et al., 1983, Science 220:868-870; Gallo, R., et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F., et al., 1986, Science 233:343-346; Guyader, M., et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. In humans, HIV replication occurs prominently in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N., et al., 1984, RNA Tumor Viruses, Weiss, R., et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439).
The HIV viral particle comprises a viral core, composed in part of capsid proteins, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated gag protein forms an outer shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M., and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish, A., et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (McDougal, J. S., et al., 1986, Science 231:382-385; Maddon, P. J., et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. While these virus:cell interactions are necessary for infection, there is evidence that additional virus:cell interactions are also required.
2.2. HIV TREATMENT
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H., et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H., et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddI, 3TC and ritonavir (Perelson, A. S., et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8+ T cells, which are essential to the control of HIV, via killer cell activity (Blazevic, V., et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of suppressive factors, notably the chemokines Rantes, MIP-1xcex1 and MIP-1xcex2 (Cocchi, F., et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10:S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith, D. H., et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R., et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O., et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R., et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533).
Recently, chemokines produced by CD8+ T cells have been implicated in suppression of HIV infection (Paul, W. E., 1994, Cell 82:177; Bolognesi, D. P., 1993, Semin. Immunol. 5:203). The chemokines RANTES, MIP-1xcex1 and MIP-1xcex2, which are secreted by CD8+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1 or HIV-2 isolates in vitro (Cocchi, F, et al., 1995, Science 270:1811-1815). These chemokines, alone or in combination, effectively suppressed the replication of several primary isolates of HIV-1, HIV-2 and SIV when tested in a variety of in vitro assays (Cocchi et al. supra). The mechanism of chemokine-mediated suppression was further delineated by a series of independent reports showing that xcex2 chemokine suppression CCR5 serves as a co-receptor for macrophage-tropic NSI isolates of HIV (Alkhatib et al., 1996, Science 272:1955; Dragic et al., 1996, Nature 381:667; Choe et al., 1996, Cell 85:1135; Berson et al., 1996, J. Virology 70:6288). However, this activity is highly specific since xcex2 chemokines blocked macrophage tropic NSI isolates but had no significant effect on T cell-tropic SI isolates of HIV-1 (Cocchi et al., supra; Alkhatib et al., supra). Thus, these and other chemokines may prove useful in therapies for some strains of HIV infection. The clinical outcome, however, of all these and other candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L., et al., U.S. Pat. No. 5,141,867; Saith, G., et al., PCT International Publication No. WO92/22,654; Shafferman, A., PCT International Publication No. WO91/09,872; Formoso, C., et al., PCT International Publication No. WO90/07,119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
2.3. LIPOPOLYSACCHARIDES
Endotoxins of gram-negative microorganisms fulfill a vital function for bacterial viability, and induce in mammalians potent pathophysiological effects. Chemically, they are lipopolysaccharides consisting of an O-specific chain, a core oligosaccharide, and a lipid component, termed lipid A. The latter determines the endotoxic activities and together with the core constituent 3-deoxy-D-manno-octulosonic acid (KDO), essential functions for bacteria.
Under normal conditions, lipopolysaccharide (LPS) is inserted in the outer surface of the outer membrane of gram negative bacteria (Schnaitman and Klena, Microbiol Rev, 57:655-682 (1993)); and Makela and Stocker, In: Handbook of endotoxin volume 1, Elsevier Biomedical Press, Amsterdam, Rietschel (ed), pp. 59-137 (1984). Complete or xe2x80x9csmoothxe2x80x9d LPS is composed of three main domains called lipid A, the O-antigen (also called the O-polysaccharide) and the core region, which creates an oligosaccharide link between lipid A and the O antigen (Schnaitman and Klena, supra; and Makela and Stocker, supra). The O-antigen is composed of oligosaccharide repeat units. The structure and number of these repeats varies depending on the bacterial species and growth conditions, typically ranging from one to fifty repeats (Schnaitman and Klena, supra; and Makela and Stocker, supra). Some bacterial generi, such as Neisseria spp., produce LPS that has low numbers of O-antigen repeats and therefore is referred to as lipoligosaccharide (LOS) simply to reflect this fact (Schnaitman and Klena, supra; and Makela and Stocker, supra).
The biological properties of LPS have been extensively investigated (Rietschel et al, FASEB J, 8:217-225 (1994); and Raetz, J Bacteriol, 175:5745-5753 (1993)). This molecule has powerful pyrogenic properties and in humans purified LPS (at doses of 200 ng to 1 xcexcg) has been shown to induce febrile responses (Greisman and Homick, J Immunol, 109:1210-1215 (1972); Greisman and Homick, J Infect Dis, 128:257-263 (1973); Abemathy and Spink, J Clin Invest, 37:219-225 (1958); Rietschel et al, supra; and Raetz, supra (1993)). These febrile responses are mediated by host proinflammatory cytokines IL-1, IL-6, and TNF-xcex1, the secretion of which is induced by LPS (Rietschel et al, supra and Raetz, supra).
The biologically active component of LPS is lipid A (Rietschel et al, supra; Verma et al, Infect Immun, 60(6):2438-2444 (1992); Alving, J Immunol Meth, 140:1-13 (1991); Alving and Richards, Immunol Lett, 25:275-280 (1990); and Richard et al, Infect Immun, 56:682-686 (1988)). Activity analysis of lipid A biosynthesis precursors or synthetic intermediates showed that various elements of lipid A are essential for pyrogenicity (Rietschel et al, supra; Raetz, supra).
For several years, it has been known that under certain circumstances, stimulation with bacterial LPS protects macrophages from HIV infection (Kornbluth et al., 1989, J. Exp. Medicine 169:1137; Bernstein et al., 1991, J. Clinical Invest. 88:540; Bagasra et al., 1992, Proc. Natl. Acad. Sci. 89:6285). LPS-mediated suppression is thought to be dependent on LPS-CD14 interactions (Bagasra et al. supra), the induction of xcex2 chemokines MIP-1xcex1, MIP-1xcex2 and RANTES (Verani et al., 1997, J. Exp. Med. 185:805) and down regulation of xcex2 chemokine receptors (Sica et al, 1997, J. Exp. Med. 185:969).
In attempts to detoxify the effects of LPS or lipid A in the treatment of Gram-negative bacteremia and septic shock, antibodies have been designed which detoxify the endotoxin activity by hydrolyzing the LPS or lipid A to products with reduced toxicity (see U.S. Pat. No. 5,597,573, issued Jan. 28, 1997).
LPS and lipid A are potent activators of pro-inflammatory cytokines, which accounts for the pyrogenic nature of these molecules. LPS has been shown to suppress HIV replication. LPS-induced suppression of HIV may be mediated through induction and/or down regulation of chemokine receptors. However, due to the toxicity of this molecule, LPS and lipid A are not viable candidates for the treatment of HIV.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present inventors have found that certain preparations containing LPS and/or lipid A variants, derivatives, and/or analogs demonstrate non-pyrogenic properties and exhibit anti-viral activities, particularly anti-HIV activities. In particular, non-pyrogenic preparations of LPS, lipid A, LPS antagonists and lipid A antagonists, and derivatives thereof induce xcex2 chemokine secretion, such as MIP-1xcex1 and MIP-1xcex2, but not proinflammatory cytokines, such as TNFxcex1, IL-1xcex2 and IL-6. The non-pyrogenic preparations of the invention, have been demonstrated by the Applicant to suppress HIV replication in human peripheral blood monocytes, as described by way of example herein. The present invention provides preparations of reduced or substantially negligible pyrogenicity of LPS variants and lipid A variants, and analogs and derivatives thereof which may be used as therapeutics for the treatment of human immunodeficiency virus infection.
The present invention also encompasses synthetic lipid A and LPS antagonists, such as, but not limited to, lipid X and lipid IVA, which suppress immunodeficiency virus replication, in particular, HIV-1 replication, and exhibit decreased induction relative to LPS and lipid A of proinflammatory cytokines such as IL-6, TNFxcex1 and IL-1xcex2. The lipopolysaccharide compositions of the present invention include those antagonists, derivatives or analogs of LPS and lipid A which exhibit reduced pyrogenicity and proinflammatory activity relative to wild-type LPS and lipid A, respectively, yet stimulate xcex2 chemokine secretion and inhibit HIV replication. The present invention fills a tremendous need for a non-toxic, long-term treatment of HIV infection and its sequelae, ARC and AIDS.
In particular, the present invention relates to LPS or lipid A preparations isolated from gram negative organisms containing at least one mutation from the group kdsA, kdsB, htrB, msbB. In a preferred embodiment of the present invention, non-pyrogenic LPS is isolated from the E. coli htrB1::Tn10 msbB::xcexa9cam double mutant MLK986. (Accession No. PTA-2794, deposited at American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 on Dec. 13, 2000 by University of Maryland Biotechnology Institute, Baltimore, Md.).
The present invention further relates to preparations of LPS or lipid A which have been differentially modified to yield reduced pyrogenicity or, preferably, substantially non-pyrogenic properties of the preparation relative to wild-type LPS and lipid A, respectively. In specific embodiments, preparations are treated by alkaline hydrolysis or acyloxyacyl hydrolase. Modified derivatives also in accordance with the present invention are derived from the group of monophosphoryl lipid A, penta-acyl lipid A, lipid IVA or lipid X. The present invention still further relates to LPS or lipid A derived from deacylation, by treatment with an alkali.
The present invention further relates to therapeutic methods and compositions for treatment and prevention of diseases and disorders associated with HIV-1 infection based on LPS or lipid A derivatives and therapeutically and prophylactically effective preparations containing a derivative of LPS or lipid A, and related analogs. Non-pyrogenic derivatives of lipid A and LPS can be identified by their failure to elicit a toxic response in mammals, their lack of proinflammatory activity, and/or their lack of induction of secretion of significant levels of pyrogenic cytokines, including IL-1xcex2, IL-6 and TNFxcex1. Preferably, non-pyrogenic derivatives are used in the therapeutic methods and compositions of the invention; alternatively, derivatives of reduced pyrogenicity relative to wild-type LPS and lipid A may be employed.
The invention provides for the treatment and prevention of HIV infection by administration of a therapeutic compound of the invention. The therapeutic compounds of the present invention include: lipid A or LPS derived from gram negative organisms containing at least one mutation selected from the group kdsA, kdsB, htrB, msbB, and derivatives and analogs of the foregoing; preparations of lipid A or LPS which have been modified to have reduced pyrogenic properties, including but not limited to, the group of monophosphoryl lipid A, penta-acyl lipid A or lipid A or LPS derivatives derived by deacylation of lipid A or LPS, treatment of LPS and lipid A with acyloxyacl hydroxylase or by treatment with an alkali, and derivatives and analogs of the foregoing. The invention also provides in vitro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of HIV. The invention also provides pharmaceutical compositions and methods of administration of therapeutics of the invention for treatment or prevention of HIV infection.
3.1. DEFINITIONS
As used herein, the following terms shall have the meanings indicated.